Biochemistry PDF Sheet 33

33 Asma’a Abu-Qtaish Leen Hawamdeh Nafiz Abutarboush Inhibitors An inhibitor, as the name implies, is a substance that interferes with the action of an enzyme and slows the rate of a reaction. Inhibitors are categorized into two types: 1. Irreversible inhibitors,(not physiological) reacts with th...

33 Asma’a Abu-Qtaish Leen Hawamdeh Nafiz Abutarboush Inhibitors An inhibitor, as the name implies, is a substance that interferes with the action of an enzyme and slows the rate of a reaction. Inhibitors are categorized into two types: 1. Irreversible inhibitors,(not physiological) reacts with the enzyme to produce a protein that is not enzymatically active and from which the original enzyme cannot be regenerated. 2. Reversible inhibitors,(might be physiological or not) can bind to the enzyme and subsequently be released, leaving the enzyme in its original condition. ❖ Mechanism-based inhibitors (Irreversible inhibitors): - Mechanism-based inhibitors mimic or participate in an intermediate step of the catalytic reaction. - The kinetic effect of irreversible inhibitors is to decrease the concentration of active enzyme → The term includes: A. Covalent inhibitors - Covalent or extremely tight bonds with active site amino acids. - Amino acids are targeted by drugs & toxins. ▪ The lethal compound [DFP] is an organophosphorus compound that served as a prototype for: o The nerve gas sarin (used in the last 10 years in Syrian war) o The insecticides malathion & parathion *How organophosphorus inhibitors work? Organophosphorus inhibitors function by covalently binding to the serine in the enzyme’s active site, making it inactive. This inhibition affects acetylcholinesterase, resulting in sustained muscle contraction. As a result, the diaphragm muscles remain contracted, leading to respiratory failure and ultimately causing death due to the inability to breathe. Remember: ACh exits the muscle contraction; however, it’s broken down by acetylcholinesterase into choline and acetate, leading to muscle relaxation. ▪ Aspirin (acetylsalicylic acid): covalent acetylation of an active site serine in the enzyme prostaglandin endoperoxide synthase (cyclooxygenase) Can’t synthesis prostaglandins or thromboxins - Aspirin resembles a portion of the prostaglandin precursor that is a physiologic substrate for the enzyme. Note: Acetate → 2 carbons - Before undergoing surgery, individuals taking aspirin are advised to discontinue its use for a specific period. This is because aspirin inhibits the COX-2 reaction, which can affect blood clotting during the surgical procedure. B. Transition state analogs - Transition state analogs & Compound that resemble intermediate stage of the reaction, These are molecules that closely mimic the transition state of a specific enzyme-catalyzed reaction. - We can't produce Transition state (Drugs cannot be designed that precisely mimic the transition state) because it's highly unstable, instead we make something very close to it, which becomes somehow similar to the substrate, so we call it substrate analog or Transition state analog. - Transition-state analogs (extremely potent inhibitors, bind more tightly) or Substrate analogs (bind more tightly than substrates). - Inhibitors that undergo partial reaction to form irreversible inhibitors in the active site are sometimes termed suicide inhibitors. Its affinity is higher than the affinity of the substrate, that’s why the enzyme bind to it tightly ▪ Penicillin (Most common used antibiotic) - A transition-state analog to glycopeptidyl transferase or transpeptidase - Required by bacteria for synthesis of the cell wall The interaction between the enzyme & Penicillin breaks the Amide bond (peptide bond) - The reaction is favored by the strong resemblance between the peptide bond in the β-lactam ring of penicillin & the transitionstate complex of the natural transpeptidation reaction. The enzyme tightly bounded to the carbonyl group, that prevent the synthesis of cell wall which leads to bacterial death. ▪ Allopurinol - A drug used to treat gout - Decreases urate production by inhibiting xanthine oxidase - The enzyme commits suicide by converting the drug to a transition-state analog - The enzyme contains a molybdenum–sulfide (Mo-S) complex that binds the substrates and transfers the electrons required for the oxidation reactions. - Xanthine oxidase oxidizes the drug allopurinol to oxypurinol, a compound that binds very tightly to a molybdenum–sulfide complex in the active site Due to its low solubility in water, urate tends to form crystals that precipitate within joints, causing painful joint inflammation. The structure of allopurinol is close to hypoxanthine, with the nitrogen and carbon positions interchanged (look at the green box). No production of urate ▪ Methotrexate - Synthetic inhibitor They have almost the same structure! - Anticancerous, nowadays it finds widespread use in the treatment of breast cancer. - Methotrexate inhibits the function of DHFR, which leads to Analog of tetrahydrofolate Tetrahydrofolate (THF) → 4 hydrogens - Binds to enzyme a 1000-fold more tightly Dihydrofolate (DHF) → 2 hydrogens - Tetrahydrofolate aids in synthesis thymidylate (TMD) which is the nucleotide form of thymidine with an additional phosphate group attached, essential for DNA synthesis and replication. When methotrexate inhibits DHFR function, it disrupts DNA synthesis by Inhibiting nucleotide base synthesis, preventing cell proliferation. This mechanism makes it effective in treating certain types of cancer. Convert DHF to THF Vitamin B9 C. Heavy metals - Tight binding of a metal to a functional group in an enzyme (Have high affinity to the active sites). - Mercury (Hg), lead (Pb), aluminum (Al), or iron (Fe) - Relatively nonspecific for the enzymes they inhibit, particularly if the metal is associated with high-dose it’ll be toxic. ▪ Mercury: binds to so many enzymes, often at reactive sulfhydryl groups in the active site - It has been difficult to determine which of the inhibited enzymes is responsible for mercury toxicity. ▪ Lead provides an example of a metal that inhibits through replacing the normal functional metal in an enzyme, such as calcium, iron, or zinc. - Its developmental & neurologic toxicity may be caused by its ability to replace Ca+2 in several regulatory proteins that are important in the central nervous system and other tissues. - Previously, lead was added to paint for improved quality, but due to its sweet taste, children were prone to ingesting it. Consequently, the use of lead in paint is now illegal in many countries. ❖Reversible inhibitors: - Characterized by a rapid dissociation of the enzyme-inhibitor complex. - Usually these inhibitors bind through non-covalent interactions & inhibitor maintains a reversible equilibrium with the enzyme. - The double-reciprocal plots are highly useful for distinguishing among these inhibitors. → Reversible inhibitors can be divided into two classes: A. Competitive inhibition - Consists of compounds very similar in structure to the substrate. In this case, the inhibitor can bind to the active site and block the substrate’s access to it. - The inhibitor competes with substrate - Increasing [S] can overcome the inhibition (to reach the same Vmax) - KM increase and Vmax remain the same, so the plot will shift to the right - Significance (ex. Hexokinase), Hexokinase catalyzes the conversion of glucose to G6P. As the concentration of G6P rises, it acts as an inhibitor by competitively binding to the active site of hexokinase. - It is important to remember that the most distinguishing characteristic of a competitive inhibitor is that substrate or inhibitor can bind the enzyme, but not both. Because both are vying for the same location, sufficiently high substrate will “outcompete” the inhibitor. This is why Vmax does not change; it is a measure of the velocity at infinite [substrate]. 1/Vmax remain the same → Y intercept remain the same -1/KM is increasing, we move toward the zero point The slope=KM/Vmax → KM The slope B. Noncompetitive inhibition - The inhibitor binds at a site other than the active site, as a result of binding, causes a change in the structure of the enzyme, especially around the active site. - The substrate is still able to bind to the active site, but the enzyme cannot catalyze the reaction when the inhibitor is bound to it. - Because the KM is a measure of the affinity of the enzyme and substrate, and because the inhibitor does not affect the binding, the KM does not change with noncompetitive inhibition. - The complex does not proceed to form product or has a lower efficiency - Vmax decrease and KM remain the same - We can decrease its influence by increasing the concentration of the enzyme 1/Vmax increase -1/KM remain the same → X intercept remain the same The slope=KM/Vmax → Vmax The slope An example helps you to distinguish between Competitive & Noncompetitive inhibitors: Let’s imagine that you have 6 blue pins, your maximum velocity to open them all = 6 pin/min. Your friend adds his red pins to yours, you start confusing between the red and the blue pins but your maximum velocity to open the blue pins remain 6 pin/min, when you add more blue pins you find it easier to reach your maximum velocity! Now your friend wanna dare your abilities, he binds your hands to see if you’ll still open the blue pins in the same velocity, unfortunately your maximum velocity decreases but you still open the 6 pins, you can re reach your original Vmax if your friend helps you! In our short story the blue pins represent the substrate, you and your friend (in the last part) represent the enzyme, the red pins were the competitive inhibitor, and finally the binding of your hands was the Noncompetitive inhibitor. I hope you will get an A in your course! THE END OF SHEET #33